File:The Biological bulletin (20385712161).jpg

Title: The Biological bulletin
Identifier: biologicalbullet189mari (find matches)
Year: [1] (s)
Authors: Marine Biological Laboratory (Woods Hole, Mass. ); Marine Biological Laboratory (Woods Hole, Mass. ). Annual report 1907/08-1952; Lillie, Frank Rattray, 1870-1947; Moore, Carl Richard, 1892-; Redfield, Alfred Clarence, 1890-1983
Subjects: Biology; Zoology; Biology; Marine Biology
Publisher: Woods Hole, Mass. : Marine Biological Laboratory
Contributing Library: MBLWHOI Library
Digitizing Sponsor: MBLWHOI Library

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Figure 2. Representative results of amplification of the repetitive region of the adhesive protein gene. Amplified products were electro- phoresed on l% agarose gel. Lanes I and 2. Mylili\ eilitlis: lanes 3 and 4, M. irossuhts: lanes 5 and 6, M. galloprovincialis. M, molecular marker (lambda DNA digested with £«>T 141). inserted into the Sma I site of pUC19. Sequences of both strands of three independent clones were determined using a 373A DNA sequencer (Applied Biosystems Inc.) and a PRISM Dyeterminator Cycle Sequencing Kit (Applied Biosystems Inc.). Results Variation in the repetitive region The primers Me 13 and Me 14 were designed to amplify the repetitive region using the sequences identical to both M. edulis and M. galloprovincialis. Since the sense primer. Me 13. corresponds to a part of the nonrepetitive region and the antisense primer. Me 14, to a part of the 3' un- translated region, the whole repetitive region is amplified by PCR. AI. edulis. M. galloprovincialis, and M. trossulus were collected at Delaware, Kamaishi, and Juneau, re- spectively. These sampling points are known to be "pure sites" at which no other species of the M. edulis complex is found (McDonald el at., 1991). We analyzed 8, 16, and 8 individuals of AI. edulis, M galloprovincialis, and M. trossulus, respectively, using primers Me 13 and Me 14. Since the repetitive region is relatively long and highly repetitive, it was difficult to amplify the whole repetitive region if the template DNA was insufficiently pure and long, but prominent bands were successfully obtained by using well-purified, high molecular weight DNA. Typical results are shown in Figure 2. Sizes of the band ranged from 2.2 to 2.8 kb. The fragments obtained from M. edulis were generally larger than those of the other two species. The sizes of bands in M. trossulus and M. galloprovincialis were similar but, on average, the former were slightly larger. Many individuals had two-banded (heterozygous) patterns, as expected for a highly variable polymorphism. One sample of M. galloprovincialis exhibited three bands, which may be a naturally occurring triploid or a mosaic individual that possesses a cell lineage having the differed length of foot protein 1 gene. It is. however, also possible that the third band is a heteroduplex of two different frag- ments. Variation in the nonrepetitive region Another set of primers. Me 15 and Me 16, was also prepared to amplify a part of the nonrepetitive region using sequences perfectly identical between AI. edulis and AI. galloprovincialis (Fig. 1). The size of the amplified fragment estimated from sequence data previously re- ported (Filpula ft a/.. 1990) is 180 bp in AI. edulis. In M galloprovincialis, the expected size is 126 bp because the sequence of M. galloprovincialis contains a deletion of 18 amino acids (Fig. 1; see also Inoue and Odo, 1994). Using these primers, 8, 32, and 16 individuals of M. edulis, M. galloprovincialis, and M. trossulus—including the same samples used in the analysis of the nonrepetitive region— were examined. PCR analysis indicated that all samples exhibited a single band. Representative results are shown in Figure 3. The position of the band was uniform in each species but differed from species to species. The size of the amplified fragments of M. edulis and M. gallopro- vincialis estimated by mobility in agarose agreed with those expected. Fragments from M. trossulus were shorter than those of M. edulis but longer than those of M. gallo- provincialis. To determine the length and sequence of the amplified fragment of M. trossulus. the band obtained from one sample (Fig. 3, Lane 3) was isolated and se- M 1 6 M
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Figure 3. Representative results of amplification of the nonrepetitive region of the adhesive protein gene. Amplified products were electro- phoresed on 4.8% NuSieve GTG agarose gel (FMC). Lanes I and 2. Mvtili.\ L'llitlfi. lanes 3 and 4, M. trosxidiix: lanes 5 and 6, M. gallopro- riiit'iuliy M, molecular marker (pUC!9 DNA digested with 7/II).

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